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. 2004 Jan;42(1):372-7.
doi: 10.1128/JCM.42.1.372-377.2004.

PCR-based genotyping of Mycobacterium tuberculosis with new GC-rich repeated sequences and IS6110 inverted repeats used as primers

Roman Kotłowski  1 , Isdore Chola Shamputa, Nabil Abdullah El Aila, Anna Sajduda, Leen Rigouts, Armand van Deun, Françoise Portaels
Affiliations

PCR-based genotyping of Mycobacterium tuberculosis with new GC-rich repeated sequences and IS6110 inverted repeats used as primers

Roman Kotłowski et al. J Clin Microbiol. 2004 Jan.

Abstract

In the present study we attempted to develop a PCR-based epidemiological tool for the differentiation of Mycobacterium tuberculosis isolates. Use of the designed primers Mtb1 (5'-CCG-GCG-GGG-CCG-GCG-G) and Mtb2 (5'-CGG-CGG-CAA-CGG-CGG-C) targeting frequently repeated 16-bp sequences in combination with primers sited at the inverted repeats flanking IS6110 allowed differentiation of M. tuberculosis isolates.

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Figures

FIG. 1.
FIG. 1.
Theoretical illustration of our genotyping method based on PCR amplification of DNA regions between two closely located IS6110 copies (A), between IS6110 and Mtb1 (B), and between IS6110 and Mtb2 (C).
FIG. 2.
FIG. 2.
Stacked histograms presenting the distribution of Mtb1 and Mtb2 sequences and the insertion sequence IS6110 in the genomes of M. tuberculosis (H37Rv and CDC1551). C, location of IS6110 at 36-nucleotide position upstream of Rv1758 and MT1805 genes coding for cutinase; DR, location of IS6110 within direct-repeat region; T, IS6110 tandem repeats.
FIG. 3.
FIG. 3.
Results of typing of 10 clones isolated from one sputum sample ITM 02-0544 from Bangladesh by three methods: IS6110-RFLP, spoligotyping, and PCR with the the primer combinations Mtb1-IS1-IS2 or Mtb2-IS1-IS2. Arrows in each of the typing methods indicate differences between clone no. 8 and nine other clones. R, M. tuberculosis Mt 14323; S, M. tuberculosis H37Rv; N, negative control; M, 100-bp DNA Ladder Plus (MBI Fermentas).
FIG. 3.
FIG. 3.
Results of typing of 10 clones isolated from one sputum sample ITM 02-0544 from Bangladesh by three methods: IS6110-RFLP, spoligotyping, and PCR with the the primer combinations Mtb1-IS1-IS2 or Mtb2-IS1-IS2. Arrows in each of the typing methods indicate differences between clone no. 8 and nine other clones. R, M. tuberculosis Mt 14323; S, M. tuberculosis H37Rv; N, negative control; M, 100-bp DNA Ladder Plus (MBI Fermentas).
FIG. 4.
FIG. 4.
Differentiation of M. tuberculosis strains by using our PCR-based genotyping method (A) and by IS6110-RFLP fingerprinting (B). Lanes: 1 and 2, M. tuberculosis ITM3288; 3 and 4, M. tuberculosis ITM3255; 5 and 6, M. tuberculosis ITM3192; 7, M. tuberculosis ITM3153; 8, M. tuberculosis ITM4454; 9, M. tuberculosis ITM3132; 10, M. tuberculosis ITM4451; 11, M. tuberculosis ITM4424; 12, M. tuberculosis ITM4337; 13, M. tuberculosis ITM4339; 14, M. tuberculosis MZ1830; 15, M. tuberculosis MZ1799; 16, M. tuberculosis KP8689; 17, M. tuberculosis KP2398; 18, M. tuberculosis KP3189; 19, M. tuberculosis SL19; 20, M. tuberculosis SL11; N, negative control; M, 100-bp DNA Ladder Plus (MBI Fermentas).
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